Projection structure and molecular architecture of OxlT, a bacterial membrane transporter

The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane 'carrier' proteins. OxlT, a representative example of the MFS, is an oxalate-transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 A resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two-fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily.     

Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function

Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the "rocker switch" may apply to certain MFS transporters, intermediate "tilted" states may exist under certain circumstances or as transitional structures. Although wet lab experimental confirmation is required, our results suggest that transport mechanisms in this transporter family should probably not be assumed to be conserved simply based on standard structural homology considerations. Furthermore, steered molecular dynamics elucidating energetic interactions of ligands with amino acid residues in an appropriately modeled transporter may have predictive value in understanding the impact of mutations and/or polymorphisms on transporter function.     

Oligomeric state of the oxalate transporter, OxlT

OxlT, the oxalate transporter of Oxalobacter formigenes, was studied to determine its oligomeric state in solution and in the membrane. Three independent approaches were used. First, we used triple-detector (SEC-LS) size exclusion chromatography to analyze purified OxlT in detergent/lipid micelles. These measurements evaluate protein mass in a manner independent of contributions from detergent and lipid; such work shows an average OxlT mass near 47 kDa for detergent-solubilized material, consistent with that expected for monomeric OxlT (46 kDa). A disulfide-linked OxlT mutant was used to verify that it was possible detect dimers under these conditions. A second approach used amino-reactive cross-linkers of varying spacer lengths to study OxlT in detergent/lipid micelles and in natural or artificial membranes, followed by analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These tests, performed under conditions where the presence of dimers can be documented for either of two known dimeric transporters (AdiC or TetL), indicate that OxlT exists as a monomer in the membrane and retains this status upon detergent solubilization. In a final test, we showed that reconstitution of OxlT into lipid vesicles at variable protein/lipid ratios has no effect on the specific activity of subsequent oxalate transport, as the OxlT content varies between 0.027 and 5.4 OxlT monomers/proteoliposome. We conclude that OxlT is a functional monomer in the membrane and in detergent/lipid micelles.     

Coupling between ATP hydrolysis and protein conformational change in maltose transporter

As the intracellular part of maltose transporter, MalK dimer utilizes the energy of ATP hydrolysis to drive protein conformational change, which then facilitates substrate transport. Free energy evaluation of the complete conformational change before and after ATP hydrolysis is helpful to elucidate the mechanism of chemical‐to‐mechanical energy conversion in MalK dimer, but is lacking in previous studies. In this work, we used molecular dynamics simulations to investigate the structural transition of MalK dimer among closed, semi‐open and open states. We observed spontaneous structural transition from closed to open state in the ADP‐bound system and partial closure of MalK dimer from the semi‐open state in the ATP‐bound system. Subsequently, we calculated the reaction pathways connecting the closed and open states for the ATP‐ and ADP‐bound systems and evaluated the free energy profiles along the paths. Our results suggested that the closed state is stable in the presence of ATP but is markedly destabilized when ATP is hydrolyzed to ADP, which thus explains the coupling between ATP hydrolysis and protein conformational change of MalK dimer in thermodynamics. Proteins 2017; 85:207–220. © 2016 Wiley Periodicals, Inc.     

Structural model for 12-helix transporters belonging to the major facilitator superfamily

The major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans. We have recently reported the three-dimensional structure, at 6.5 A resolution, of the oxalate transporter, OxlT, a representative member of this superfamily. In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane. Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins. Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily.     

Probing structure-function relationships and gating mechanisms in the CorA Mg2+ transport system

Recent crystal structures of the CorA Mg(2+) transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg(2+)-selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg(2+) selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.     

Three-dimensional structure of a bacterial oxalate transporter

The major facilitator superfamily (MFS) represents one of the largest classes of evolutionarily related membrane transporter proteins. Here we present the three-dimensional structure at 6.5 A resolution of a bacterial member of this superfamily, OxlT. The structure, derived from an electron crystallographic analysis of two-dimensional crystals, reveals that the 12 helices in the OxlT molecule are arranged around a central cavity, which is widest at the center of the membrane. The helices divide naturally into three groups: a peripheral set comprising helices 3, 6, 9 and 12; a second set comprising helices 2, 5, 8 and 11 that faces the central substrate transport pathway across most of the length of the membrane; and a third set comprising helices 1, 4, 7 and 10 that participate in the pathway either on the cytoplasmic side (4 and 10) or on the periplasmic side (1 and 7). Overall, the architecture of the protein is remarkably symmetric, providing a compelling molecular explanation for the ability of such transporters to carry out bi-directional substrate transport.     

Cysteine scanning mutagenesis of TM5 reveals conformational changes in OxlT, the oxalate transporter of Oxalobacter formigenes

We constructed a single-cysteine panel encompassing TM5 of the oxalate transporter, OxlT. The 25 positions encompassed by TM5 were largely tolerant of mutagenesis, and functional product was recovered for 21 of the derived variants. For these derivatives, thiol-directed MTS-linked agents (MTSEA, MTSCE, and MTSES) were used as probes of transporter function, yielding 11 mutants that responded to probe treatment, as indicated by effects on oxalate transport. Further study identified three biochemical phenotypes among these responders. Group 1 included seven mutants, exemplified by G151C, displaying substrate protection against probe inhibition. Group 2 was comprised of a single mutant, P156C, which had unexpected behavior. In this case, we observed increased activity if weak acid/base or neutral probes were used, while exposure to probes introducing a fixed charge led to decreased function. In both instances, the presence of substrate prevented the observed response. Group 3 contained three mutants (e.g., S143C) in which probe sensitivity was increased by the presence of substrate. The finding of substrate-protectable probe modification in groups 1 and 2 suggests that TM5 lies on the permeation pathway, as do its structural counterparts, TM2, TM8, and TM11. In addition, we speculate that substrate binding facilitates TM5 conformational changes that allow new regions to become accessible to MTS-linked probes (group 3). These biochemical data are consistent with the recently developed OxlT homology model.     

Protonation of Glu135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

Major facilitator superfamily (MFS) transporters typically need to alternatingly sample the outward-facing and inward-facing conformations, in order to transport the substrate across membrane. To understand the mechanism, in this work, we focused on one MFS member, the L-fucose/H⁺ symporter (FucP), whose crystal structure exhibits an outward-open conformation. Previous experiments imply several residues critical to the substrate/proton binding and structural transition of FucP, among which Glu¹³⁵, located in the periplasm-accessible vestibule, is supposed as being involved in both proton translocation and conformational change of the protein. Here, the structural transition of FucP in presence of substrate was investigated using molecular-dynamics simulations. By combining the equilibrium and accelerated simulations as well as thermodynamic calculations, not only was the large-scale conformational change from the outward-facing to inward-facing state directly observed, but also the free energy change during the structural transition was calculated. The simulations confirm the critical role of Glu¹³⁵, whose protonation facilitates the outward-to-inward structural transition both by energetically favoring the inward-facing conformation in thermodynamics and by reducing the free energy barrier along the reaction pathway in kinetics. Our results may help the mechanistic studies of both FucP and other MFS transporters.     

Role of aromatic localization in the gating process of a potassium channel

Position of the transmembrane aromatic residues of the KirBac1.1 potassium channel shifts from an even distribution in the closed state toward the membrane/solute interface in the open state model. This is the first example of an integral membrane protein making use of the observed preference for transmembrane aromatic residues to reside at the interfaces. The process of aromatic localization is proposed as a means of directing and stabilizing structural changes during conformational transitions within the transmembrane region of integral membrane proteins. All-atom molecular dynamics simulations of the open and closed conformers in a membrane environment have been carried out to take account of the interactions between the aromatic residues and the lipids, which may be involved in the conformational change, e.g., the gating of the channel.     

Role of Aromatic Localization in the Gating Process of a Potassium Channel

Position of the transmembrane aromatic residues of the KirBac1.1 potassium channel shifts from an even distribution in the closed state toward the membrane/solute interface in the open state model. This is the first example of an integral membrane protein making use of the observed preference for transmembrane aromatic residues to reside at the interfaces. The process of aromatic localization is proposed as a means of directing and stabilizing structural changes during conformational transitions within the transmembrane region of integral membrane proteins. All-atom molecular dynamics simulations of the open and closed conformers in a membrane environment have been carried out to take account of the interactions between the aromatic residues and the lipids, which may be involved in the conformational change, e.g., the gating of the channel.     

Visualization of SV2A conformations in situ by the use of Protein Tomography

The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography™. We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins.     

Crystal structures of the maltodextrin/maltose-binding protein complexed with reduced oligosaccharides: flexibility of tertiary structure and ligand binding

The structure of the maltodextrin or maltose-binding protein, an initial receptor for bacterial ABC-type active transport and chemotaxis, consists of two globular domains that are separated by a groove wherein the ligand is bound and enclosed by an inter-domain rotation. Here, we report the determination of the crystal structures of the protein complexed with reduced maltooligosaccharides (maltotriitol and maltotetraitol) in both the "closed" and "open" forms. Although these modified sugars bind to the receptor, they are not transported by the wild-type transporter. In the closed structures, the reduced sugars are buried in the groove and bound by both domains, one domain mainly by hydrogen-bonding interactions and the other domain primarily by non-polar interactions with aromatic side-chains. In the open structures, which abrogate both cellular activities of active transport and chemotaxis because of the large separation between the two domains, the sugars are bound almost exclusively to the domain rich in aromatic residues. The binding site for the open chain glucitol residue extends to a subsite that is distinct from those for the glucose residues that were uncovered in prior structural studies of the binding of active linear maltooligosaccharides. Occupation of this subsite may also account for the inability of the reduced oligosaccharides to be transported. The structures reported here, combined with those previously determined for several other complexes with active oligosaccharides in the closed form and with cyclodextrin in the open form, revealed at least four distinct modes of ligand binding but with only one being functionally active. This versatility reflects the flexibility of the protein, from very large motions of interdomain rotation to more localized side-chain conformational changes, and adaptation by the oligosaccharides as well.     

Crystal structure of group II chaperonin in the open state

Thermosomes are group II chaperonins responsible for protein refolding in an ATP-dependent manner. Little is known regarding the conformational changes of thermosomes during their functional cycle due to a lack of high-resolution structure in the open state. Here, we report the first complete crystal structure of thermosome (rATcpnβ) in the open state from Acidianus tengchongensis. There is a ～30° rotation of the apical and lid domains compared with the previous closed structure. Besides, the structure reveals a conspicuous hydrophobic patch in the lid domain, and residues locating in this patch are conserved across species. Both the closed and open forms of rATcpnβ were also reconstructed by electron microscopy (EM). Structural fitting revealed the detailed conformational change from the open to the closed state. Structural comparison as well as protease K digestion indicated only ATP binding without hydrolysis does not induce chamber closure of thermosome.     

Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes

An OxlT homology model suggests R272 and K355 in transmembrane helices 8 and 11, respectively, are critical to OxlT-mediated transport. We offer positive evidence supporting this idea by studying OxlT function after cysteine residues were separately introduced at these positions. Without further treatment, both mutant proteins had a null phenotype when they were reconstituted into proteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treated with MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively charged amino group. In each case, there was a 2-fold increase in the Michaelis constant (K(M)) for oxalate self-exchange (from 80 to 160 microM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V(max) compared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEA introduced the desired modification. We also examined substrate selectivity for the treated derivatives. While oxalate remained the preferred substrate, there was a shift in preference among other substrates so that the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate > formate > malonate). This shift is consistent with the idea that the substrate-binding site is reduced in size via introduction of the SCH(2)CH(2)NH(3)(+) adduct, which generates a side chain that is approximately 1.85 A longer than that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essential components of the OxlT substrate-binding site.     

Conformational equilibria and free energy profiles for the allosteric transition of the ribose-binding protein

The ribose-binding protein (RBP) is a sugar-binding bacterial periplasmic protein whose function is associated with a large allosteric conformational change from an open to a closed conformation upon binding to ribose. The crystal structures of RBP in open and closed conformations have been solved. It has been hypothesized that the open and closed conformations exist in a dynamic equilibrium in solution, and that sugar binding shifts the population from open conformations to closed conformations. Here, we study by computer simulations the thermodynamic changes that accompany this conformational change, and model the structural changes that accompany the allosteric transition, using umbrella sampling molecular dynamics and the weighted histogram analysis method. The open state is comprised of a diverse ensemble of conformations; the open ribose-free X-ray crystal conformations being representative of this ensemble. The unligated open form of RBP is stabilized by conformational entropy. The simulations predict detectable populations of closed ribose-free conformations in solution. Additional interdomain hydrogen bonds stabilize this state. The predicted shift in equilibrium from the open to the closed state on binding to ribose is in agreement with experiments. This is driven by the energetic stabilization of the closed conformation due to ribose-protein interactions. We also observe a significant population of a hitherto unobserved ribose-bound partially open state. We believe that this state is the one that has been suggested to play a role in the transfer of ribose to the membrane-bound permease complex.     

Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating

Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.     

Diversity of conformational states and changes within the EF-hand protein superfamily

The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca2+ binding, is found in a large number of functionally diverse Ca2+ binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca2+ to the open conformational state in its presence. It is now clear from structures of other EF-hand proteins that this "closed-to-open" conformational transition is not the sole model for EF-hand protein structural response to Ca2+. More complex modes of conformational change are observed in EF-hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF-hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF-hand proteins and its correlation with target recognition.     

Conformational flexibility of the leucine binding protein examined by protein domain coarse-grained molecular dynamics

Periplasmic binding proteins are the initial receptors for the transport of various substrates over the inner membrane of gram-negative bacteria. The binding proteins are composed of two domains, and the substrate is entrapped between these domains. For several of the binding proteins it has been established that a closed-up conformation exists even without substrate present, suggesting a highly flexible apo-structure which would compete with the ligand-bound protein for the transporter interaction. For the leucine binding protein (LBP), structures of both open and closed conformations are known, but no closed-up structure without substrate has been reported. Here we present molecular dynamics simulations exploring the conformational flexibility of LBP. Coarse grained models based on the MARTINI force field are used to access the microsecond timescale. We show that a standard MARTINI model cannot maintain the structural stability of the protein whereas the ELNEDIN extension to MARTINI enables simulations showing a stable protein structure and nanosecond dynamics comparable to atomistic simulations, but does not allow the simulation of conformational flexibility. A modification to the MARTINI-ELNEDIN setup, referred to as domELNEDIN, is therefore presented. The domELNEDIN setup allows the protein domains to move independently and thus allows for the simulation of conformational changes. Microsecond domELNEDIN simulations starting from either the open or the closed conformations consistently show that also for LBP, the apo-structure is flexible and can exist in a closed form.

ATP-driven MalK dimer closure and reopening and conformational changes of the "EAA" motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2)

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK(2)) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.